Evaluation of the acute toxicity and 28‐days subacute toxicity of the alcoholic extract from Ganoderma leucocontextum

Abstract Ganoderma leucocontextum is a well‐known traditional medicine in Tibet Autonomous Region, which has benefits, such as anti‐hypoxia, neurotrophic action on nerves, easing coughs and relieving asthma, strengthening the body and prolonging life. However, few research have focused on its negative effects, possibly jeopardizing its safety. The purpose of this study is to evaluate the acute and subacute toxicity of an alcoholic extract from G. leucocontextum (GLA) in vivo. The phytochemical characterization analysis showed that alcoholic extract from G. leucocontextum were rich in polysaccharides, triterpenoids. Then, in acute oral toxicity, male and female mice from Institute of Cancer Research (ICR) were orally administered with 16 g/kg GLA and were observed for 14 days. In the subacute toxicity, male and female Sprague–Dawley (SD) rats were orally administered with 2, 4, and 8 g/kg doses of GLA for 28 days. There was no death or clinical changes in male and female mice in the acute toxicity test. During the subacute toxicity test, the difference in body weights, food consumption, biochemical and hematological parameters, and organ coefficients between treated and control groups were unrelated to GLA treatment. The obtained data show that the GLA had no significant toxic effects when administered orally to male and female rats in acute and subacute toxicity.

. According to the Tibetan medical classic Shel Gong Shel Phreng, G. leucocontextum was beneficial for treating hypoxia, trophic action on the nerves, easing cough, relieving asthma, strengthening the body, and prolonging life .
Because of its ethnopharmacological significance, investigations into the pharmacological potential and phytochemical analyses of this species have been conducted. G. leucocontextum included more polysaccharides and triterpenoids than other Ganoderma variants, and G. leucocontextum contained novel polysaccharides and triterpenoids Gao et al., 2020Gao et al., , 2021. Scientific evidence has demonstrated that the triterpenoids from G. leucocontextum have anticancer (Li et al., 2019;Liu et al., 2018;Zhao et al., 2016), hypolipidemic effects Wang et al., 2015;Zhang et al., 2018), antidiabetics Zhang et al., 2017), and neuroprotective effects . GLP-1 and GLP-3, polysaccharides from G. leucocontextum, have potent antioxidant and immune boosting effects (Gao et al., 2020(Gao et al., , 2021. The potential toxicity of G. leucocontextum has been overlooked, and the safe dose has yet to be determined. The toxicity of G. leucocontextum aqueous extract was previously assessed (Pan et al., 2021). In the subacute toxicity, no toxicity was shown at the highest dose of 3 g/kg. However, the possible toxicity of other medicinal parts of G. leucocontextum was ignored in this previous study, and the design of toxicology studies did not follow the regulatory requirements (China Food and Drug Administration, 2014;OECD, 2002OECD, , 2008. The possibility of harm at other concentrations or in alternative types of extracts is not ruled out by these statistics. The recommended daily dosage of G. leucocontextum was 6-12 g for adults (60 kg of body weight), but there have not been any essential scientific studies to support it yet (Sichuan Medical Products Administration, 2020). Furthermore, there are no safety studies on the acute or subacute toxicity of G. leucocontextum alcoholic extracts, implying that more research is needed to determine the extracts' toxicity. The purpose of this research was to determine the toxicity of G. leucocontextum alcoholic extracts in mice and rats using acute and subacute toxicity tests.

| Plant materials and extraction
The fruiting bodies of G. leucocontextum were collected in Linzhi, Tibet, in July 2020, and were identified by associate Prof. Bengui Ye, leucocontextum . In summary, the dried G. leucocontextum powder was soaked in 70% ethanol at a 1:15 (w/v) ratio for 24 h before being refluxed three times at 100°C. The extracted solution was filtered and concentrated under vacuum, and an alcoholic extract of G. leucocontextum (GLA, 1466 g) was obtained after freeze-drying. The w/w extraction rates of GLA were 14.66%, respectively. In other words, 1 g of GLA rough extract equals 6.82 g of raw material.

| Phytochemical characterization
Triterpenes and polysaccharides are the major active components of G. leucocontextum, hence their quantity in GLA was measured. The content of polysaccharides and total triterpenoids present in GLA was quantified using TU-1810 UV/Vis (PERSEE, China) and methods specified in the literature . The anthrone-sulfuric acid method was used to quantify the content of polysaccharides, with anhydrous glucose as the reference standard (y = 4.3719x + 0.0049, R = .9991). The vanillin-ice acetic acid method was used to measure total triterpenoids, with oleanolic acid as the reference standard (y = 10.605x − 0.0045, R = .9992).
The phytochemicals contained in the GLA were analyzed using a Dionex Ultimate 3000 UHPLC linked to a Q-Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, America). The findings were assessed using the Xcalibur data system and Compound Discoverer 3.1 software after samples were dissolved in methanol (1 g/ml) and run entirely at a range of 200-800 m/z (Thermo Fisher Scientific, America). The experiments were conducted in the C-18 column (Thermo Scientific, America). The first mobile phase (0.1% formic acid) and the second mobile phase (methanol) were on a gradient elution of 0 min: 50% of B, 0-2 min: 50% of B, 3 min: 78% of B, 9 min: and 82% of B, 11 min: 87% of B, 14 min: and 95% of B, 21 min: 100% of B, 23 min: and 50% of B (end of run), at the flow rate of 0.26 ml/min. The identification of peaks was performed by comparing the present results with the retention times (Rt) and mass spectrums from the software library.

| Test animals
From Chengdu Dossy Experimental Animals CO., LTD.
(SCXK2020-030), we purchased 40 male and female ICR (Institute of Cancer Research) mice weighing 18-22 g, and 80 male and female Sprague-Dawley (SD) rats aged 3 weeks, weighing 50-60 g. All animals were kept in standard laboratory conditions, with 12 h of light/ darkness, a relative temperature of 22 ± 2°C, and a humidity of 40-60%. They were provided with food and water at random (Jensen et al., 2013). Five days before the experiment, these animals were acclimatized to the experimental conditions.

| Acute toxicity test
Because G. leucocontextum has been used in folklore for a long time, it was assumed to be a nontoxic or low-toxic ethnic medicine, and an acute oral toxicity test was performed using the limit test according to the procedure outlined in National Standards of Food Safety (GB 15193.3-2014). As a result, the experiments were carried out at the maximum concentration and volume of these extracts.
Twenty male and twenty female ICR mice were randomly divided into two experimental groups: the GLA-treatment group (16 g/kg GLA) and the negative control group, with 10 male and 10 female mice in each group. Mice in both groups fasted for 12 h before administration to eliminate gastrointestinal feed and drank freely, and then received 16 g/kg GLA via gavage all at once. Three hours after gavage, the diet was resumed, and body weights, general state, development indicators, and toxic reactions were continuously recorded. At the same time, the negative control group was given saline and recorded. After the experiment, all of the animals were fasted for 12 h, anesthesia was administered with 3% sodium pentobarbital, and blood was collected for hematological analysis and serum biochemistry. The CA470 Blood Coagulator Analyzer was used to determine hemoglobin (HGB), hematocrit (HCT), white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) (MEDONIC, Sweden). A Chemistry Analyzer AU400 was used to measure albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (UREA), creatinine (CREA), alkaline phosphatase (ALP), and total cholesterol (CHOL) (Olympus, Tokyo, Japan). Pathological examinations were then performed on the heart, liver, spleen, lung, and kidney. Animals that have been poisoned or euthanized should be dissected as soon as possible to preserve the abnormal organs.

| 28-days subacute toxicity test
Because G. leucocontextum was nontoxic (LD 50 > 16 g/kg) in the acute oral toxicity test, the high-dose group should be increased as much as possible in the subacute toxicity test without affecting the animals' feeding and nutritional balance (Xu et al., 2021). In accordance with the dose design requirements of National Food Safety Standards (GB 15193.22-2014) and the results of the acute toxicity test, the highdose group's dose should be increased as much as possible under the presumption that it will not affect the animals' feeding and nutritional balance, and the interval of the decreasing dose should be 2-4 times.
After 5 days of environmental adaptation, 80 SD rats were randomly divided into four groups: high-dose (8 g/kg), medium-dose (4 g/kg), low-dose (2 g/kg), and negative control (0 g/kg). Each group was divided into half males and half females. Saline solutions of GLA with concentrations of 400 mg/ml (maximum solubility), 200, and 100 mg/ ml were prepared using saline as the solvent, and the negative control group was given an equal volume of saline with an intragastric volume of 20 ml/kg by weight. Urine samples were collected from each rat at the end of day 28 dosing for analysis. All rats were fasted for 12 h before being anesthetized with 3% sodium pentobarbital and blood was collected in ethylenediaminetetraacetic acid tubes, sodium citrate anticoagulation tubes, and non-anticoagulated tubes for hematological analysis, and serum biochemistry.

| Mortality and clinical signs
During the experiment, the animals were observed twice a day for their coat, skin, eyes, mucous membranes, secretions, excretions, respiratory system, voluntary activities, and behavioral performance. The animals' general clinical manifestations, duration of poisoning, and death were all documented. Weak animals should be isolated, and dead animals should be dissected as soon as possible. Every 3 days, the body weight of each rat was measured and recorded, and the feed consumption of each group was calculated.

| Hematological analysis
At the end of the experiment, all rats were fasted for 12 h and blood was obtained from the abdominal aorta under anesthesia for blood index examination.

| Urine examination
The day before the test, urine was collected for 24 h from each rat using a metabolic cage, and the color of the urine was recorded. A N-400 Urine analyzer was used to perform qualitative analysis of urine protein, specific gravity, pH, glucose, bilirubin, and occult blood (Dirui Industrial Co., Ltd., Jilin, China).

| Macroscopic examination and organ weights
Following blood collection, all rats were subjected to a gross autopsy, which included examination of the body surface, cranial, thoracic, and abdominal cavities, organs, and the location, morphology, color, and size of organs with the naked eye. The organ/body ratio was calculated by weighing the absolute weights of the heart, thymus, adrenal, liver, kidney, spleen, testes, and ovaries. The relative weight of each organ was calculated according to the equation below (Equation [1]).

| Histopathological examination
At dissection, the brain, heart, liver, spleen, lung, kidney, pancreas, testis, and ovary were collected from negative control and GLAtreated animals of each group and fixed in 4% paraformaldehyde fix solution. After fixation, all tissues and organs were dehydrated, embedded, sectioned, stained using hematoxylin and eosin, and observed under a microscope (Xiang et al., 2015).

| Statistical analysis
These values are expressed as the means ± standard deviation, and one-way vascular analysis was performed using SPSS 26.0 statistical software (SPSS Inc., Chicago, IL, USA). A one-way analysis of variance was performed, followed by a t-test to assess differences between groups. All treated groups were compared with the control group, * represented p < .05, ** represented p < .01.

| Phytochemical characterization
The content of total polysaccharides is 15.74 mg of anhydrous glucose equivalent/g of GLA (1.57%), and the content of total triterpenoids is 63.13 mg of oleanolic acid equivalent/g of GLA (6.31%).
The retention time (RT) and molecular weight (MW) of the retention peak of the LC-MS were analyzed to identify the phytochemicals.
In the present study, the LC/MS chromatogram showed the retention peaks of about 41 compounds. As shown in Figure 1, it was inferred that GLA contains a variety of alkane acids and triterpenes (Table S1).

| Acute oral toxicity test
During the 14-day observation period, all male and female ICR mice in the negative control and GLA-treated groups survived. No animals displayed any negative effects or clinical symptoms of poisoning during the study. The body weight change, hematological parameters and serum biochemistry parameters of treated mice were normal (Tables S2-S4). Figure 2 shows that the mice in the GLA-treated group did not have abnormal pathological examinations of the major organs after autopsies (heart, liver, spleen, lung, and kidney).

| Mortality and clinical signs
No adverse effects or fatalities were observed during the 28-day oral GLA treatment (2, 4 and 8 g/kg). Furthermore, when compared to the negative control group, the GLA-treated animals showed no abnormalities in their behavior or voluntary activity, general behavior, coats, glandular production, breathing, or fecal characteristics. Figure 3 shows the mean body weights of female and male rats. Female rats in the 2 g/kg/d GLA-treatment group gained significantly more weight from days 21 to 24 when compared with the negative control group (0 g/kg/d) (p < .05). Over the period of the experiment, no additional significant differences were found between the treatment groups and the negative control group in both genders. All groups had no effect on the food consumption of male and female rats. In addition, the food consumption of 2 g/kg within male rats decreased in a short time. Nevertheless, the changes were minimal, related to a single time point, and had no toxicological relevance. (Figure 4).

| Hematological analysis
In hematological analysis, the results indicated that the WBC in male SD rats was significantly increased at the dose of 4 g/kg/d of GLA (p < .05). In females, the LPR of the 8 g/kg/d group decreased significantly when compared with that in the negative control group (p < .05), and the PLT was significantly increased in the highdose group (p < .05). None of the other hematological parameters showed a difference in significance between groups in either gender (Table S5).

| Serum biochemistry
Serum biochemistry analysis data are shown in Table S6. In males and females, the level of ALB was significantly increased at the dose of 8 g/kg of GLA groups (p < .05). In males, CREA and ALP were significantly increased in mid-dose (4 g/kg/d) treatment group (p < .05) after 28 days of treatment, compared with the negative control group. In females, the level of ALT decreased significantly at the dose of 2 g/kg/d of GLA (p < .05). The significant decrease (p < .05) of CK and A/G were observed in high-dose groups after 28 days of treatment, the level of CL − increased significantly at the doses of (1) Relative organ weight = Absolute organ weight (g) ∕ Body weight (g) * 100 % 8 g/kg of GLA groups (p < .05). After 28 days of GLA administration, the other serum biochemistry parameters did not differ from the control group.

| Urine examination
In the qualitative urine analysis, there were no obvious differences in urobilinogen, bilirubin, ketone bodies, urine blood, protein, nitrite, leukocytes, glucose, specific gravity, pH, and vitamin C in both female and male rats following a 28-day oral administration period of GLA (data not shown).

| Organ coefficient
After 28 days of treatment with GLA, organ weights were shown in Table S7. In comparison to the control group, the high-dose GLAtreated group's kidney relative weight in females was significantly higher (p < .05). There were no other significant changes in relative organ weights in any of the treatment groups when compared with the control group. Furthermore, a comprehensive examination of all the major organs of the rats confirmed no abnormalities.

| Histopathological analysis
Pathological analysis of male and female rats' organs at the end of the 28-day experiment revealed no detectable abnormalities in the tissues of the brain, heart, liver, spleen, lung, kidney, testis, or ovary.
Histopathological examination revealed no abnormalities in microscopic examination in the control or high-dose groups, and no serious histopathological problems in the high-dose group, as shown in In the acute oral toxicity test, ICR mice were fed 16 g/kg/day of GLA without any side effects or deaths, and at necropsy, no serious organ abnormalities were found. Based on the circumstances of this study, both in male and female rats, the median lethal dose of GLA was predicted to be greater than 16 g/kg body weight. Substances with an oral LD50 greater than 5 g/kg may be considered harmless (Kennedy Jr. et al., 1986). According to the criteria of acute toxic classifications of Ministry of Health, China, 2014, the GLA is actually not toxic (Xu et al., 2021).
In the 28-day subacute oral toxicity study, GLA caused neither any sign of toxicity nor death in SD rats. Changes in body weight have been used to predict pharmacological and chemical side effects. GLA had the effect of increasing the food consumption of female rats. The body weight of female rats in the low-dose group was faster than that of the negative control group during the whole experiment period. The body weight and food intake statistical findings showed no specific dosage association or sex responsiveness, indicating that the increases in body weight and food consumption were not thought to have any clinical significance.
The hematological function is amongst the most sensitive indications of the toxicity of drugs, analyzing changes in blood and biochemical parameters are essential (Xiang et al., 2015). Hematological parameters in the treated groups showed alterations, such as a minor increase in WBC, PLT, and a decline in LPR in the 8 g/kg female rats. In histopathological analysis, GLA did not cause any abnormalities in the blood metabolism-related organs (liver, spleen, or kidney). The alterations were considered to be toxicologically unconnected because they did not depend on dosage or were not reflected by changes in other critical parameters and were within normal physiological limits (Piao et al., 2013;Villas Boas et al., 2018;Zhong-Ze et al., 2010).
The liver is the primary organ involved in the metabolism of xenobiotics, assessing its function is critical in recognizing the potential toxicity of the therapies administered. Albumin is a significant biomarker of liver function, hypoalbuminemia could be caused by the liver producing less, losing more, or increasing proteolysis and clearance. ALB and A/G showed a decreasing trend in male and female rats at high doses, while ALT and AST did not show abnormalities and no albumin was detected in the urine. No abnormalities in the gastrointestinal tract, liver or kidney were found in the pathological examination. In addition, the changes were unrelated to dose and were within the laboratory's historical standard range of controls. These findings show that rats administered 8 g/kg of GLA did not experience liver or renal damage. Therefore, feeding GLA at 8 g/kg/d was not responsible for the decrease in serum levels of ALB and A/G. The liver plays a central role in detoxification. Female CK, ALT, and AST levels have decreased, which could be attributed to the presence of triterpenes and polysaccharides in GLA Oluwafemi Adetuyi et al., 2020;Tong et al., 2020).
Increasing in creatinine is typically associated with visible damage to functional nephrons, which is a good indicator of renal function. There were statistically significant differences in CREA in 4 g/ kg GLA-treated animals when compared with the control group. The predominant histological findings of the kidney in the rats at 4 g/kg were normal glomerular size and number, with no tubular abnormalities. These findings suggested that the renal function might not be damaged by GLA at 4 g/kg. The changes in CL − in the female groups F I G U R E 4 Mean food consumption of male and female Sprague-Dawley rats treated with the alcoholic extract from Ganoderma leucocontextum during the 28day toxicological assessment F I G U R E 3 Mean body weight of male and female Sprague-Dawley rats treated with the alcoholic extract from Ganoderma leucocontextum during the 28-days toxicological assessment were not considered treatment-related because they showed no apparent dose dependence, and they were also within the normal range of laboratory reference data.
In relative organ weight, the changes in kidney relative organ weight were likewise inconsistent between the sexes and did not follow any dose-related patterns. Additionally, no kidney histological abnormalities were found. Therefore, the slight reduction in kidney weight observed in males at 8 g/kg was not deemed harmful. In the histopathological examination, all of these pathological alterations were intermittently found during the histological analysis in both the controls and the GLA-administered rats; however, neither sex consistently displayed any of these changes. As a result, these lesions may be regarded as spontaneous or accidental in origin but unrelated to the therapy of GLA.

| CON CLUS IONS
The results of an acute study showed that the oral median lethal dose (LD 50 ) of the alcoholic extract from G. leucocontextum is more than 16 g/kg body weight. The subacute toxicity test, which was orally administered with 2, 4, and 8 g/kg doses of GLA for 28 days, revealed no significant organ and tissue changes, the no-observedadverse-effect level (NOAEL) for GLA was 8 g/kg/day. The NOAEL level of GLA in the present study is equated to an intake for humans of 8.6 g/kg (raw material), which is 43 times the recommended daily allowance for adults. According to the study, G. leucocontextum was deemed safe for medical use. This research extended the G. leucocontextum's safe range, served as a springboard for later studies on the activity of the plant's various medicinal parts, and established a theoretical foundation for G. leucocontextum in the homologies of medicine and food, as well as a scientific foundation for future research.

ACK N OWLED G M ENTS
The authors thank Tibet Education Department and Tibet Science and Technology Department for financial support (Project number 2020QT022) (Project number XZ202101ZD0021G).

CO N FLI C T O F I NTE R E S T
The authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.

I N FO R M E D CO N S E NT
All the listed authors have read and approved the submitted manuscript.